In our previous studies it has been demonstrated that phosphorylation of the neuron specific intermediate filament proteins, such as neurofilaments (NFs) were tightly regulated developmentally and topographically, and generally confined to the axonal compartment and selectively occurs on proline directed serine (Ser) and threonine (Thr) residues. It was recognized that in neurodegenerative disorders such as Alzheimers disease (AD) and Amyotrophic lateral sclerosis (ALS), the pathology was characterized by an accumulation of aberrantly phosphorylated cytoskeletal proteins in perikarya on these residues, suggesting that topographic regulation had been compromised. Neurodegenerative diseases including AD are complex and chronic disorders that involve the disruption of the neuronal network in the human brain. We have made first time the comprehensive study of quantitative mass spectrometric analysis of human neuron specific major cytoskeletal proteins, medium (NF-M)and high (NF-H) molecular weight neurofilament proteins from control adult and AD matched brains. This study aimed to extend our knowledge of specific phosphorylation sites to provide insight into the role of kinases and phosphatases in the formation of neurofibrillary tangles (NFTs) and aggregates. The quantitative mass spectroscopic technique ( iTRAQ) used in this study identified specific phosphorylation sites among multiple lysine serine/threonine proline (KS/TP) repeats and non-S/TP sites in NF-M/H. For example, evidence of phosphorylation of proline-directed and non-proline-directed Ser residue suggests the involvement of both proline-directed and non-proline-directed kinases in AD.The iTRAQ analysis revealed primarily that specific Ser/Thr sites are phosphorylated at a higher fold in AD brain compared with control brain and that not all the neurofilament KSP sites are phosphorylated at similar levels in AD. Some of them are phosphorylated at a greater abundance compared with other sites. Hipocampal,CA1 pyramidal cells are known for susceptibility to neurofibrillary degeneration in AD. In human brain, these neurons also show an age-related perikaryal accumulation of NFs. Most of the phosphorylation sites in AD tangles are perikaryonal constituents. Normal perikarya do not contain phosphorylated neurofilament proteins.Our immunohistochemistry data suggest that phospho-NF is predominantly detected in intracellular NFTs and extracellular plaques in AD. The iTRAQ data obtained with phosphorylated NF-M from AD brain also show the higher abundance of phosphorylation of non-proline-directed Ser or Thr residues,suggesting the involvement of also non-proline-directed kinases in addition to proline - directed kinases in AD brain. This study first time demonstrates the importance of regulation of NF-M phosphorylation in physiology and pathology. It is found that KSP repeat phosphorylation of NF-M is significantly higher compared with NF-H, despite the greater number of these sites in NF-H. We were able to obtain the sequence coverage for 13/18 phosphorylation sites of NF-M;however, iTRAQ analysis resulted in the aberrant phosphorylation of 10/43 phosphorylation sites of NF-H in AD brain. Based on the stoichiometry, there is 2-fold higher KSP repeats phosphorylated in NF-M compared with NF-H;therefore, we believe that NF-M might contribute more to aberrant phosphorylation in AD compared with other cytoskeletal proteins. and also suggests that specific antibodies could be generated to the SP and non-SP sites that are phosphorylated at a greater abundance and can be used as bio-markers in AD and the specific inhibitors of these kinase can be used as therapeutic reagents for AD and other neurodegenerative diseases involved in aberrant neuronal cyytoskeletal protein phosphorylation. We have began to study the role of proein phosphatases to further understand the molecular basis of the phosphorylation during aging and associated neuropathology. We found that the protein phospatase activity declined relatively more, rather than activation of kinases, underlies aging-related neuronal cytoskeletal proten hyperphosphorylation. Purified protein phosphatase (PP2A) dephosphorylated the heavy neurofilament (NF-H) subunit or its extensively phorphorylated carboxyl-terminal domain in vitro. In cultured primary hippocampal neurons, inhibiting PP2A induced NF-H phosphorylation without activating known neurofilament kinases. Neurofilament phosphorylation in the mouse CNS, as reflected by levels of the RT-97 antibody phosphoepitope associated with late axon maturation, more than doubled during the 12-month period after the NF-H phosphorylation plateaued at P21. This was accompanied by declines in levels and activity of PP2A but not PP2B, and no rise in activities of neurofilament kinases (Erk1,2, cdk5 and JNK1,2). Inhibiting PP2A in mice in vivo restored brain RT-97 to levels seen in young mice. Declining PP2A activity, therefore, can account for rising neurofilament phosphorylation in maturing brain, potentially compounding similar changes associated with adult-onset neurodegenerative diseases.